RNA Polymerase II CTD Tyrosine 1 is Required for Efficient Termination by the Nrd1-Nab3-Sen1 Pathway (NET-Seq)

Transcription termination is key to gene regulation as it prevents transcription interference with neighboring genes. In Saccharomyces cerevisiae, termination at protein-coding genes is coupled to the cleavage of the nascent transcript, while most non-coding RNA transcription relies on a cleavage-independent termination pathway involving Nrd1, Nab3 and the helicase Sen1 (NNS pathway). In both pathways, the recruitment of termination factors involves phosphorylated forms of the RNA polymerase II C-terminal domain (CTD) but the contribution of individual CTD residues was never systematically investigated. Here, we investigated the impact of mutating phosphorylation sites in the CTD on termination. We observed widespread termination defects at protein-coding genes in mutants for Ser2 or Thr4 but rare defects in Tyr1 mutants for this class of genes. Instead, mutating Tyr1, or its phosphatase Glc7, led to widespread termination defects at non-coding genes known to terminate via the NNS pathway. These defects can be suppressed by slowing down transcription, suggesting that Tyr1 mediates termination via the regulation of elongation or pausing. Our work redefines the role of Tyr1 in termination at protein-coding genes in budding yeast and highlights its key role in termination by the NNS pathway. NET-seq experiments of WT RNAPII and an RNAPII mutant where the CTD is mutated such that all 26 repeats contain an Y1F substitution. Cells express either Flag-tagged WT RPB1 or Flag-tagged Y1F mutant RPB1 on a centromeric plasmid under the control of the RPB1 promoter. The endogenous RPB1 gene is Myc-tagged. NET-seq experiments were performed using an anti-Flag antibody in duplicate, for a total of four samples.